Project Abstract Our long-term goal is to establish a methodology to efficiently deliver proteins to the cytosol of live mammalian cells. Current delivery systems such as cell-penetrating peptides (CPPs) are inefficient because they promote extensive endosomal entrapment and degradation of their protein cargo. This results in experimental artifacts that render the proper imaging of protein biosensors impractical. We propose to solve this problem by optimizing the ability of CPPs to selectively disrupt endosomal membranes so as to achieve a more efficient release of the protein from endosomes into the cytosol and reduce degradation. Our specific aims are to: 1) identify the conditions required for optimal CPP-mediated protein delivery, 2) evaluate and optimize novel CPP systems designed to efficiently disrupt membranes upon acidification of the lumen of endosomes, 3) define the relations between endosomal pH, CPP concentration and endosomal release activity of CPPs. To achieve these goals, we will use a recently developed imaging technique to unambiguously measure the endocytic and cytosolic distribution of a protein probe delivered into live cells. Novel protein probes that can report on the properties of endosomes containing CPP-protein conjugates will also be developed. We anticipate that our results will provide key chemical insights in the critical step of endosomal disruption and lay a firm foundation for the rational design of efficient delivery systems that can achieve cytosolic targeting of protein biosensors with low background of untargeted protein. This will not only enable the microscopy of live cells with externally administered imaging probes but should have an important impact on the entire field of delivery of cell-impermeable macromolecules in general.